Recombinant human hemoglobin: Modification of the polarity of the .beta.-heme pocket by a valine67(E11) .fwdarw. threonine mutation
| Autor: | J K O'Donnell, Enrico Bucci, Zygmunt Gryczynski, William S. Brinigar, Janusz Kowalczyk, Clara Fronticelli, John S. Olson |
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| Rok vydání: | 1993 |
| Předmět: |
Threonine
Hemeprotein Macromolecular Substances Stereochemistry Molecular Sequence Data Mutant Bohr effect Heme Ligands Biochemistry Hemoglobins chemistry.chemical_compound Escherichia coli Humans Amino Acid Sequence Cloning Molecular Binding site Binding Sites Base Sequence Chemistry Valine Oligonucleotides Antisense Recombinant Proteins Kinetics Mutagenesis Site-Directed Hemoglobin Mathematics Oxygen binding |
| Zdroj: | Biochemistry. 32:1235-1242 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi00056a006 |
| Popis: | Using the mutagenesis and a gene expression system previously described [Fronticelli et al. (1991) J. Protein Chem. 10, 495-501], we have replaced Val67E11 in the distal heme pocket of the beta-chains of hemoglobin with Thr. The valine to threonine substitution is isosteric and only modifies the polarity of the beta-heme environment. The absorption and CD spectra of the resultant mutant hemoglobin were essentially the same as that of wild-type protein, indicating that the mutation did not cause any large conformational changes and that a water molecule was not coordinated to the ferrous iron atom. Equilibrium measurements of oxygen binding to the mutant indicate a 2-fold decrease in overall affinity relative to native or wild-type human hemoglobin. Thermodynamic analyses of O2 binding curves, based either on the sequential Adair model or on the MWC two-state model, indicated that the overall decrease of O2 affinity in the system was due to a lower association equilibrium constant for the intermediates of oxygenation, particularly those involved at the third ligation step. The functional characteristics of the mutant hemoglobin in either the T- or R-state were not modified greatly by the mutation; however, the Bohr effect and sensitivity to C1- were increased, suggesting a role of the intermediates of oxygenation in the modulation of these parameters.(ABSTRACT TRUNCATED AT 250 WORDS) |
| Databáze: | OpenAIRE |
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